The zona pellucida, a glycocalyx that surrounds the mammalian oocyte, is comprised of three sulfated glycoproteins designated, ZP-1, ZP-2 and ZP-3. After secretion, these proteins are formed into an extracellular matrix which appears to have three major functions: (1) mediation of species-specific sperm binding associated with ZP-3 (2) blocking of post-fertilization polyspermy associated with ZP-2 and (3) protection of the growing embryo as it passes down the oviduct. We have developed and characterized a battery of monoclonal antibodies specific for the zona proteins. Five bind exclusively to ZP-2, three bind both to ZP-2 and ZP-3 and an additional two cell are specific for ZP-3 alone. Each anti-ZP-2 monoclone binds to 1.3 x 10tothe8 sites (roughly 2% of the available ZP-2) which by indirect immunofluorescence appears distributed in a fibrous network throughout the zona pellucida. Although the zona proteins are not linked covalently, laser photo-bleaching showed that ZP-2 has no lateral diffusion. Furthermore, at the resolution of light microscopy the distribution of ZP-2 is not modified during pre-implantation although the functions of the zona pellucida change substantially. Passive immunization of mice with anti-ZP-2 monoclonal antibodies dramatically inhibits sperm binding to oocyte but does not affect other functions of the zona pellucida. The monoclonal antibody localizes to the zona surrounding growing oocytes in the ovary. After ovulation, the bound antibody prevents the formation of the 2-cell embryo. This effect is reversible after 70 days and the resumption of fertility is associated with the disappearance of anti-zona antibody from the ovary. Although ZP-3 is thought to be the sperm receptor, two monoclonal antibodies specific for ZP-2 effectively blocked fertilization. Thus, the monoclonal antibodies appear to block sperm binding by steric hinderance. Fluorescent and immuno-electron microscopic studies are in progress using two differentially labeled monoclones each specific to ZP-2 or to ZP-3. From these investigations we hope to better understand the supramolecular structure of the zona pellucida and how it changes during early development.